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A biosensor for rapid and simultaneous visual identification of high-risk human papillomavirus (HPV) genotypes 16 and 18 in clinical samples based on polymerase chain reaction (PCR) integrated lateral flow strip platform was developed. Using an one-step protocol to extract nucleic acid rapidly and the functionalized primer sets specific to HPV-16 and 18 were designed for the simultaneous amplification. In the presence of target HPV genotypes, the corresponding functionalized primer sets will participate in the PCR process and produce numerous duplex functionalized dsDNA amplicons. With the bridge effect of duplex functionalized dsDNA amplicons between gold nanoparticles-fluorescein isothiocyanate antibody conjugates (AuNP-FITC antibody conjugates) and other two antibodies on corresponding test line (T

In this study, we demonstrated the role of whey protein isolate (WPI)/(-)-epigallocatechin-3-gallate (EGCG) covalent conjugates/gellangum double network emulsion gels by duo-induction of glucono-δ-lactone (GDL) and CaCl

We describe an isothermal, single-reaction, and one-step method for signal-on quantification of terminal deoxynucleotidyl transferase (TdT) activity based on the periodic elongation and assembly of polythymine embedded activatable molecular beacon (PTA-MB) into DNA nanostructures. PTA-MB is easily designed according to the rule of the conventional molecular beacon (MB) but engineered with a polyT composed loop. Upon exposure to the specific target TdT, the MB is first elongated with an adenine-rich (A-rich) long chain so that it can then act as the anchoring substrate to capture many original PTA-MBs along its strand. Their unfolding contributes to preliminary fluorescence emission. Significantly, the assembled PTA-MBs can also be elongated and hybridized with residual free PTA-MBs for the second round of signal amplification. Accordingly, multiple rounds of elongation, assembly, and activation of initial PTA-MBs can lead to the formation of DNA nanostructures and induce a dramatically enhanced fluorescence signal for qualitative and quantitative evaluation of TdT activity. The final assay indicated a limit of detection (LOD) of 0.042 U mL−1 TdT and showed excellent selectivity for TdT versus other common enzymes. Moreover, the practical applicability was validated by direct/absolute quantification of TdT in real biological specimens and accurate monitoring of the activity of TdT pretreated by low/high temperature and heavy metal ions. These findings demonstrated that this functional PTA-MB and its unique assembly behavior is most likely to promote the study of oligonucleotide probe-based DNA assembly, providing a reliable, convenient, and universal platform for precise and point-of-care monitoring of various biomolecules.

Detection of pathogenic bacteria is of vital significance for combating and preventing infectious diseases. In this work, we developed a multivalent aptamer probe (Multi-VAP)-based trigging isothermal circular amplification (TICA) for rapidly and ultrasensitively detecting

Single base mutations detection is crucial for the diagnosis and treatment of cancer. However, the current methods with poor selectivity and sensitivity required large instruments, which are difficult to meet clinical demands. Herein, we develop a CRISPR/Cas9 based visual colorimetric platform to specifically detect all single base mutations. In this strategy, the Recombinase Polymerase Amplification (RPA) was firstly used to amplify the target, and introduced the PAM site in the target DNA sequence by designing the point mutation primer, thus achieving detection for all single base mutations by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated specific recognition. With the help of CRISPR/Cas9 system, those RPA products can release single strand DNA to hybridize with the padlock probe and trigger rolling circle amplification (RCA). Based on the magnetic separation, HRP-gold nanoparticles complex (hGNPs) and biotin modified probe (Bio-probe) were further used to achieve enhanced visual variations assay by hybridizing with RCA products. Benefiting from the RPA assisted triple signal amplification, this method not only showed enhanced sensitivity with a limit of detection (LOD) as low as 0.2 fM and 0.01% of KRAS-G12D mutation percentage, but the specificity against KRAS-G12D mutation also be synergistically enhanced by combining the CRISPR/Cas9-mediated specific recognition with the specific T4 ligation reaction of RCA system. Furthermore, this system has been successfully used to visually detect genome in serum, suggesting its great potential for point-of-care diagnosis in clinical.

Rapid authentication of meat adulteration is of great importance for food safety. A new FRET‐based self‐signal‐on fluorescent colorimetric PCR approach for rapid, sensitive and specific identification of horsemeat ingredient has been developed. A specific hairpin probe was designed and integrated with the forward primer for the detection of horsemeat. This specific hairpin sequence was designed to generate fluorescent signal only when primers are incorporated into the formation of double‐stranded PCR product. The fluorescent signal of the PCR product is in a linear relationship with the proportion of horsemeat ingredient, and could be used for the quantitative detection of horsemeat ingredient in meat products without any post‐treatment or analysis of the amplicons. Based on the fluorescent signal intensity, rapid and convenient detection of horsemeat ingredient in beef products was successfully achieved with satisfied specificity and sensitivity.

Rapid, accurate, and effective authentication of unlabeled meat components in commercial meat products is of great significance for guaranteeing food safety and human health. In this research, we have constructed a single-strand binding protein (SSB)-assisted performance improved fluorescence polarization (FP) protocol for authentication of chicken component in meat products. The SSB-assisted FP strategy involves the use of fluorescein isothiocyanate (FITC)-labeled primers to perform polymerase chain reaction (PCR). In addition, FP enhancement was accomplished by mixing the amplified product with SSB. Based on the designed mechanism, the free primers in the final amplified product can be tightly bound by SSB, which greatly restricts the rotation of FITC, thereby resulting a high FP signal. In the presence of chicken DNA, the transformation of single-stranded primers to double-stranded amplicons greatly hinders the combination of SSB and primers, leading to the decrease of FP signal. This method well couples the advantages of both PCR and FP technology. Through this new strategy, as low as 0.035% (wt. %) of chicken adulteration in meat samples can be well authenticated. Moreover, this method is also available for commercial meat products. We expect this SSB-assisted FP strategy will provide new insights into the construction of versatile analysis tools in the field of food safety detection.

The authors describe a rapid and low-cost approach for multiplex microRNA(miRNA) assay on lateral flow nucleic acid biosensor (LFNAB). The principle of assay is based on sandwich-type nucleic acid hybridization reactions to produce gold nanoparticle (GNP)-attached complexes (ssDNA-microRNA-ssDNA/GNPs), which are captured and visualized on the test zone of LFNAB. By designing three different test zones on LFNAB, simultaneous detection of microRNA-21, microRNA-155 and microRNA-210 was achieved with an adding-measuring model by using GNP as visual tag. The method was challenged by testing the microRNAs in spiked serum samples with satisfied results. In our perception, the test is a particularly valuable tool for clinical application and biomedical diagnosis, particularly in limited resource settings.

Fluorescence polarization (FP) signal is a self-referencing fluorescence signal, and it is less dependent on dye concentration and environmental interferences, which makes FP measurement a highly attractive alternative sensing technology to conventional fluorescent detection methods. Here we adopted a strategy for rapid increase of molecular weight to increase the FP signal for the detection of meat adulteration. The molecular weight of fluorescent labeled primers increased rapidly by slight preamplification, and the FP values were varied accordingly. We found a positive correlation between adulteration ratio and the FP signals. Detection limit for adulterated beef can be reached as low as 0.1% (wt %), meeting or better than the most detection requirements. On the basis of this proposed amplification-integrated FP method, both the standard samples and the commercial processed beef samples were successfully authenticated with satisfied results.

Due to the similar clinical symptoms of influenza (Flu) and coronavirus disease 2019 (COVID-19), there is a looming infection threat of concurrent Flu viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we introduce a customized isothermal amplification integrated lateral flow strip (LFS) that is capable performing duplex reverse transcription-recombinase polymerase amplification (RT-RPA) and colorimetric LFS in a sequential manner. With customized amplification primer sets targeted to SARS-CoV-2 (opening reading frame 1a/b and nucleoprotein genes) and Flu viruses (Flu A and Flu B), the platform allows the rapid and simultaneous visual screening of SARS-CoV-2 and Flu viruses (Flu A and Flu B) without cross reactivity, false positives, and false negatives. Moreover, it maximally eases the detection, reduces the detection time (1 h), and improves the assay performance to detect as low as 10 copies of the viral RNA. Its clinical application is powerfully demonstrated with 100% accuracy for evaluating 15 SARS-CoV-2-positive clinical samples, 10 Flu viruses-positive clinical samples, and 5 negative clinical samples, which were pre-confirmed by standard qRT-PCR. We envision this portable device can meet the increasing need of online monitoring the serious infectious diseases that substantially affects health care systems worldwide.

A target triggered dual-DNA machine, consisting of one rolling circle amplification (RCA)-based circular DNA machine (RCA-CDM) and one cyclical strand displacement amplification (CSDA)-based bidirectional DNA machine (CSDA-BDM), was developed for robust miRNA determination.

Analysis of molecular species is needed for applications in diagnosis of infections and genetic diseases. Herein, we demonstrate a target DNA-responsive ultrahigh fluorescence signal-on DNA amplification system via periodically programmed building and collapse of DNA networks. In this system, a pair of oligonucleotides of padlock probe (PP) and palindromic hairpin probe (PHP) are utilized. The presence of target DNA firstly hybridizes with PP, allowing the occurrence of rolling circle amplification (RCA) to produce RCA products with tandem repeats in abundance to bind and unfold numbers of PHPs. The conformational change of PHPs enables the building of DNA networks via the intermolecular palindrome pairing, but then makes the DNA networks collapsed via the palindrome-induced strand displacement polymerization. The displaced RCA products are dynamically reused to undergo periodically programmed multiple rounds of DNA network building and collapse. Depend on the bidirectional DNA assembly and disassembly, a strikingly amplified fluorescence can be collected to ultrasensitive and specific detection of target DNA. The practicability has been demonstrated by evaluating target-spiked human serum, saliva, and urine samples with acceptable recoveries and reproducibility. Therefore, this newly explored method opens a promising avenue for the detection of nucleic acids with low abundance in biochemical analysis and diseases diagnosis.

We propose a visual strategy for simultaneous detection of multiple adulterated components in beef by integration of multiple polymerase chain reaction (mPCR) with the lateral flow strip (LFS). The primer sets for adulterated components are uniquely designed with different nucleic acid tags (NAT), enabling the amplicons with specific wobbled sequences at two opposite ends. The wobbled sequences will precisely hybridize with the pre-immobilized capture probes on T lines (T1, T2 and T3) and C line, contributing to the coloration of LFS. Taking advantages of extraordinary amplification efficiency of PCR and simplicity of LFS, common adulterated components including chicken, duck and pork can be easily detected with LOD as low as 0.01% (wt%), which is comparable to that of quantitative real-time polymerase chain reaction (qPCR) but with more simplified operations and reduced costs. The method can be extended to identification of other components by replacing the functional primer set. This method can be a useful candidate for meat quality control at the resource-limited setups.

A novel high-sensitivity authentication method has been demonstrated for the rapid visual detection of adulterated meat based on both the lateral flow strip (LFS) platform and on polymerase chain reaction (PCR). After the rapid extraction of genomic components from meat, the on-site amplification of the target DNA of adulterated duck meat is carried out with the rationally designed functional FITC- and biotin-modified primer set, thereby producing numerous double-stranded DNA (dsDNA) products dually labelled with FITC and biotin. The FITC-labelled terminal end of the products binds to the pre-immobilized FITC antibody on the test line of the strip, and the biotin-labelled terminal end binds to the streptavidin-conjugated gold nanoparticles, resulting in a visible test line on the LFS for the rapid identification of duck meat in adulterated beef. After optimization, an adulteration ratio as low as 0.05% can be easily measured, which is more sensitive than other common adulteration authentication methods and is even comparable to instrumental methods. Moreover, 22 commercial processed meat samples were tested with this new strategy, and 4 adulterated samples were successfully identified by both the classic method and our method. In essence, the present authentication method is simple in design, convenient in operation, and can be easily extended to the identification of other adulteration components just by replacing the modified primers.